Tryptophan determination of food proteins by h.p.l.c. after alkaline hydrolysis

Abstract

Abstract The alkaline hydrolysis of proteins prior to tryptophan analysis has been evaluated, and a method for the measurement of tryptophan by reverse phase h.p.l.c. after alkaline hydrolysis has been proposed and compared with published methods. Hydrolysis with either LiOH or NaOH gave similar results. Tryptophan values and the recovery of added 5‐methyltryptophan were similar when hydrolysis was made under tap‐water vacuum (1500 Pa) or at high vacuum (2 Pa). Partially‐hydrolysed starch reduced the losses of tryptophan during hydrolysis better than thiodiglycol. High levels of indolyl‐3‐propionic acid or 5‐methyltryptophan did not further improve tryptophan recovery. 5‐Methyltryptophan is the preferred internal standard; its recovery after hydrolysis more closely resembled that of protein‐bound tritiated tryptophan from goat casein than did the recoveries of α‐methyltryptophan or free 14 C‐tryptophan. Under some conditions, the recovery of protein bound tryptophan was lower than the recovery of free tryptophan. The stability of tryptophan in hydrolysates was improved by using a phosphate buffer pH 7.0 containing 0.02% sodium azide.